Despite the significant developments of recombinant DNA techniques, there is an increasing demand for biological products obtained by cultivation of mammalian cells. Such cells are used for physiological and biochemical cell studies, as well as for the production of various virus vaccines, hormones, biochemicals, interferons, monoclonal antibodies, plasminogen activators, and the like.
Many tissue cells require a solid surface for growth and proliferation (so-called anchorage-dependent cells). In production scale it has been common to cultivate such cells on the inside of rotating bottles. In order to achieve sufficient cell amounts several hundreds of such bottles are used in a batch. This production system is cumbersome and labour and material consuming, and a further drawback is that it in practice is impossible to monitor and control the process.
An improvement of the solid surface cultivation technique is the use of growth surfaces in the form of so-called "microcarriers", i.e. small (150-250 micron diameter) bead-shaped particles of porous or solid polymers such as cross-linked dextran. By using the microcarrier technique there is obtained an improved ratio between the surface area available for cell growth and the total volume of the culture. For example, in a one liter bottle 3 g of microcarrier particles can provide a surface area of about 18,000 cm.sup.2, whereas the inside of the same bottle only provides a growth area of about 400-500 cm.sup.2, which means an increase of the available surface area by a factor of about 40. The microcarrier technique is used commercially for the production of virus vaccines.
Although the problem of available surface area as a limiting factor seems to have been solved by the microcarrier technique, there are other factors limiting the scale-up of the mammalian cell cultures. Of these limiting factors the problem of oxygen transfer is considered to be the most critical one. Thus, the cells require a steady supply of oxygen, and a linear relationship between oxygen demand and cell concentration is typically observed. Because of the proteins of the growth medium, bubble aeration would lead to severe foaming, and oxygen is therefore commonly supplied by surface aeration.
Microcarrier cultures have to be stirred for keeping the microcarriers in suspension. The density of typical microcarriers is about 1.03. The cell wall of mammalian cells is much thinner than the cell wall of microorganisms, which means that tissue cells are more sensitive to shearing forces. The number of collisions between the microcarriers increases with increasing stirring intensity. Because of these factors the cell yield increases with decreasing stirring intensity, and the ideal stirring intensity (in this respect) is the one at which the microcarriers are just about kept in suspension. This requirement on a comparatively low stirring seems to be the main reason for the insufficient supply of oxygen to the culture in surface aeration.
It has been proposed (Fleischaker R. J. et al, Oxygen Demand and Supply in Cell Culture, European J. Upl. Microbiol. Biotechnol. 12 (1981), p. 193-197) to solve the problem of supplying sufficient amounts of oxygen to cell cultures by providing thin-walled silicone tubing at the bottom of the cell cultivation vessel. However, also this proposal has been found to be inadequate for large scale operation. For example, about 30 m of 2.5 cm silicone tubing would be required for oxygenating a 1000 1 batch of HeLa cells.
A summary of the state of the art in mammalian cell culture and the problems encountered in the scaling-up thereof has been made by M W Glacken et al in Trends in Biotechnology, Vol. 1, No. 4, 1983, p. 102-108, "Mammalian cell culture: engineering, principles and scale-up", which is incorporated herein by reference.